The present invention relates to a method for counting leukocytes and an apparatus for counting leukocytes. In particular, the present invention relates to a method and an apparatus suitable to count leukocytes in a platelet preparation or an erythrocyte preparation.
Platelet preparations and erythrocyte preparations are mainly used for alleviation of thrombocytopenia and anemia, surgical operations and so forth. Considering side effects and the like, it is not desirable from a viewpoint of quality that leukocytes are present in a platelet preparation or an erythrocyte preparation. Thus, the number of leukocytes that can be contained in a small amount in a platelet preparation or an erythrocyte preparation is measured for quality control.
Usually, the leukocyte count in a platelet preparation or an erythrocyte preparation is measured by baring nuclei of leukocytes and staining them. That is, leukocytes are accumulated by a centrifuge or the like, stained and then placed in a Nageotte chamber (hemocytometer) so that observers visually count the number using a microscope. Since platelets are rarely dissolved in this method, however, leukocytes are buried in the platelets, which results in deteriorated measurement accuracy. In addition, visual measurement is extremely inefficient. Furthermore, in this measurement method, observers often contact blood preparations with a possibility of biohazard (biological contamination). Therefore, a safe method that achieves automatization and facilitation of the measuring operation as well as improvement of measurement accuracy is presently desired.
On the other hand, in general, nuclei of leukocytes must be bared to stain the leukocytes for measurement. It has been known that a surfactant is added for this purpose. However, no method for counting leukocytes has been known, wherein a cytolytic agent that bares nuclei of leukocytes and solubilizes platelets or erythrocytes is used to solubilize platelets or erythrocytes in a platelet preparation or an erythrocyte preparation.
The present invention has been accomplished in the light of the above circumstances. The object of the present invention is to provide a method and an apparatus for readily measuring leukocyte count in a platelet preparation or an erythrocyte preparation.
As a result of the present inventors"" efforts to achieve the aforementioned object, they have been found that measurement of the leukocyte count can be facilitated and a measurement apparatus without requiring visual measurement can be obtained by utilizing a cytolytic agent that can bare nuclei of leukocytes and solubilize platelets or erythrocytes, because such a cytolytic agent can bare nuclei of leukocytes and solubilize platelets or erythrocytes when it is added to a platelet preparation solution or an erythrocyte preparation solution. Thus, the present invention has been accomplished.
That is, the present invention provides a method for counting leukocytes in a platelet preparation by staining the leukocytes, comprising adding a cytolytic agent capable of baring nuclei of leukocytes and solubilizing platelets to a solution of the platelet preparation to bare nuclei of the leukocytes and solubilize platelets in the solution of the platelet preparation.
In the present specification, terms xe2x80x9cplatelet preparationxe2x80x9d and xe2x80x9csolution of the platelet preparationxe2x80x9d are used. As for these terms, if a platelet preparation is originally in the form of a solution, xe2x80x9cplatelet preparationxe2x80x9d is equivalent to xe2x80x9csolution of the platelet preparationxe2x80x9d. It is also contemplated that, even if a platelet preparation is in the form of a solid or the like, the preparation can be used as a solution after dissolution.
The present invention also provides a method for counting leukocytes in a platelet preparation by staining the leukocytes, comprising:
mixing and shaking a solution of the platelet preparation solution, a cytolytic agent capable of baring nuclei of leukocytes and solubilizing platelets and a dye, in an accumulation container comprising an opening, a sidewall portion and a bottom portion, a part or all of the sidewall portion having a horizontal sectional area gradually increasing in a direction from the bottom portion towards the opening, to solubilize platelets, bare nuclei of the leukocytes and stain the leukocytes,
setting the accumulation container on a centrifuge to accumulate the stained leukocytes on the bottom portion of the accumulation container, and
counting the stained leukocytes.
In measurement by baring nuclei of leukocytes and staining the leukocytes, what is actually measured is usually DNA aggregates of stained bared nuclei of individual leukocytes. In the present specification, the term xe2x80x9cleukocytesxe2x80x9d may be used to refer not only to leukocytes in the normal state, but also to the DNA aggregates of stained bared nuclei of leukocytes.
In the above method for counting leukocytes in the platelet preparation, the cytolytic agent added to the solution of the platelet preparation is preferably selected from the group consisting of anionic surfactants, cationic surfactants, amphoteric surfactants and nonionic surfactants. The amount of the cytolytic agent added to the solution of the platelet preparation solution is preferably 0.2 to 5% (w/v).
The present invention also provides a method for counting leukocytes in a platelet preparation by staining the leukocytes, comprising:
placing a solution of the platelet preparation in an accumulation container comprising an opening, a sidewall portion, and a bottom portion having a membrane filter through which leukocytes are impassable, a part or all of the sidewall portion having a horizontal sectional area gradually increasing in a direction from the bottom portion towards the opening,
filtering the solution of the platelet preparation through the membrane filter provided at the bottom portion of the accumulation container containing the solution of the platelet preparation to accumulate the leukocytes on the bottom portion,
adding a surfactant and a dye to the leukocytes accumulated on the bottom portion to bare nuclei of the leukocytes and stain the leukocytes, and
counting the stained leukocytes.
The present invention also provides a method for counting leukocytes in an erythrocyte preparation by staining the leukocytes, comprising adding a cytolytic agent capable of baring nuclei of leukocytes and solubilizing erythrocytes to a solution of the erythrocyte preparation to bare nuclei of the leukocytes and solubilize erythrocytes in the solution of the erythrocyte preparation.
In the present specification, terms xe2x80x9cerythrocyte preparationxe2x80x9d and xe2x80x9csolution of the erythrocyte preparationxe2x80x9d are used. As for these terms, if an erythrocyte preparation is originally in the form of a solution, xe2x80x9cerythrocyte preparationxe2x80x9d is equivalent to xe2x80x9csolution of the erythrocyte preparationxe2x80x9d. It is also contemplated that, even if an erythrocyte preparation is in the form of a solid, the preparation can be used as a solution after dissolution.
The present invention also provides a method for counting leukocytes in an erythrocyte preparation by staining the leukocytes, comprising:
mixing and shaking a solution of the erythrocyte preparation, a cytolytic agent capable of baring nuclei of leukocytes and solubilizing erythrocytes and a dye, in an accumulation container comprising an opening, a sidewall portion and a bottom portion, a part or all of the sidewall portion having a horizontal sectional area gradually increasing in a direction from the bottom portion towards the opening, to solubilize erythrocytes, bare nuclei of the leukocytes and stain the leukocytes,
setting the accumulation container on a centrifuge to accumulate the stained leukocytes on the bottom portion of the accumulation container, and
counting the stained leukocytes.
In the above method for counting leukocytes in the erythrocyte preparation, the cytolytic agent is preferably selected from the group consisting of anionic surfactants, cationic surfactants, amphoteric surfactants and nonionic surfactants. The amount of the cytolytic agent added to the solution of the erythrocyte preparation is preferably 0.1 to 10% (w/v).
The present invention also provides a leukocyte accumulation container comprising an opening, a bottom portion and a sidewall portion, a part or all of the sidewall portion having a horizontal sectional area gradually increasing in a direction from the bottom portion towards the opening. The present invention also provides such a leukocyte accumulation container wherein the bottom portion has a membrane filter through which leukocytes are impassable. The maximum diameter of the bottom portion of the accumulation container according to the present invention is preferably 0.2 to 5 mm. The maximum diameter of the bottom portion means the longest diameter irrespective of the shape of the bottom portion. For example, if the bottom portion has a circular shape, the diameter of the circle is the maximum diameter. If it has a quadrangular shape, the length of the diagonal is the maximum diameter.
The present invention also provides an apparatus for counting leukocytes comprising:
any one of the above leukocyte accumulation containers having an opening, a bottom portion and a sidewall portion, a part or all of the sidewall portion having a horizontal sectional area gradually increasing in a direction from the bottom portion towards the opening,
a lens portion for projecting the state of the bottom portion of the leukocyte accumulation container as an image of which magnification can be changed by the lens portion,
detection means for detecting the number of the leukocytes accumulated on the bottom portion of the leukocyte accumulation container by analyzing the image of the bottom portion of the leukocyte accumulation container projected via the lens portion, and
output means for outputting detection results obtained by the detection means,
wherein the detection means comprises an image-capturing portion having an image-capturing surface for capturing an image of the bottom portion of the leukocyte accumulation container projected via the lens portion, an image analysis processor that identifies leukocytes in the image of the bottom portion of the leukocyte accumulation container on the image-capturing surface and a counter for leukocyte count, and
the bottom portion of the leukocyte accumulation container has a size such that the image of the entire bottom portion is in the image-capturing surface of the detection means as one image. The image-capturing portion preferably comprises CCD image-processing means.
The present invention will be described in detail below.